Metal complexes of N-heterocyclic carbenes

ABSTRACT

The present invention generally relates to metal complexes of N-heterocyclic carbenes that contain one or more additional active moieties and/or groups therein. In one embodiment, the present invention relates to metal complexes of N-heterocyclic carbenes that contain an anti-fungal and/or anti-microbial moiety and/or group in combination with one or more additional active moieties and/or groups selected from fluoroquinolone compounds or derivatives thereof; steroids or derivatives thereof; anti-inflammatory compounds or derivatives thereof; anti-fungal compounds or derivatives thereof; anti-bacterial compounds or derivatives thereof; antagonist compounds or derivatives thereof; H 2  receptor compounds or derivatives thereof; chemotherapy compounds or derivatives thereof; tumor suppressor compounds or derivatives thereof; or C 1  to C 16  alkyl heteroatom groups where the heterotatom is selected from S, O, or N. In still another embodiment, the present invention relates to metal complexes of N-heterocyclic carbenes that contain an anti-fungal and/or anti-microbial moiety and/or group in combination with two or more additional active moieties and/or groups selected from fluoroquinolone compounds or derivatives thereof; steroids or derivatives thereof; anti-inflammatory compounds or derivatives thereof; anti-fungal compounds or derivatives thereof; anti-bacterial compounds or derivatives thereof; antagonist compounds or derivatives thereof; H 2  receptor compounds or derivatives thereof; chemotherapy compounds or derivatives thereof; tumor suppressor compounds or derivatives thereof; or C 1  to C 16  alkyl heteroatom groups where the heterotatom is selected from S, O, or N.

RELATED APPLICATION DATA

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/482,410, filed on Jul. 7, 2006, which is acontinuation-in-part application of U.S. patent application Ser. No.10/569,563, filed on Nov. 13, 2006, which is a 35 U.S.C. §371application of International Application No. PCT/US2004/029285, filed onSep. 7, 2004, which claims priority to U.S. Provisional PatentApplication No. 60/500,737, filed on Sep. 5, 2003. Additionally, thisapplication claims priority to U.S. Provisional Patent Application No.61/250,795, filed on Oct. 12, 2009, and entitled “Metal Complexes ofN-Heterocyclic Carbenes.” All of these patent applications areincorporated herein in their entireties by reference.

The present invention was made in the course of research that wassupported by Award Number NIH R15 CA 96739-01; and Award Number NSFCHE-0116041. The United States government may have certain rights to theinvention or inventions herein.

FIELD OF THE INVENTION

The present invention generally relates to metal complexes ofN-heterocyclic carbenes that contain one or more additional activemoieties and/or groups therein. In one embodiment, the present inventionrelates to metal complexes of N-heterocyclic carbenes that contain ananti-fungal and/or anti-microbial moiety and/or group in combinationwith one or more additional active moieties and/or groups selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N. In still another embodiment, the present inventionrelates to metal complexes of N-heterocyclic carbenes that contain ananti-fungal and/or anti-microbial moiety and/or group in combinationwith two or more additional active moieties and/or groups selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N.

BACKGROUND OF THE INVENTION

Silver has long been used for its antimicrobial properties. This usagepredates the scientific or medical understanding of its mechanism. Forexample, the ancient Greeks and Romans used silver coins to maintain thepurity of water. Today silver is still used for this same purpose byNASA on its space shuttles. Treatment of a variety of medical conditionsusing silver nitrate was implemented before 1800. A one percent silvernitrate solution is still widely used today after delivery in infants toprevent gonorrheal ophthalmia. Since at least the later part of thenineteenth century, silver has been applied in a variety of differentforms to treat and prevent numerous types of bacteria relatedafflictions.

Other treatments, such as the application of silver foil to postsurgical wounds to prevent infection survived as a medical practice intothe 1980's in Europe, and silver nitrate is still used as a topicalantimicrobial agent. In the 1960's the very successful burn treatmentsilver complex, silver sulfadiazine, shown in Formula 1 below, wasdeveloped. Commercially known as Silvadene® Cream (one percent) thiscomplex has remained one of the most effective treatments for preventinginfection of second and third degree burns. Silver sulfadiazine has beenshown to have good antimicrobial properties against a number ofgram-positive and gram-negative bacteria. It is believed that the slowrelease of silver at the area of the superficial wound is responsiblefor the process of healing. Studies on surgically wounded rats haveshown the effectiveness of both silver nitrate and silver sulfadiazineto aid in the healing process. By using these common silverantimicrobial agents, inflammation and granulation of wounds werereduced, although the complete mechanism for these phenomena is notunderstood.

Recently developed silver-coating techniques have lead to the creationof a burn wound dressing called Acticoat. The purpose of this dressingis to avoid adhesion to wounds while providing a barrier againstinfection. Some clinical trials have also demonstrated the ease ofremoval of the dressing in contrast to conventional wound dressingstreated with silver nitrate. Acticoat has shown an increase inantibacterial function over both silver nitrate and silver sulfadiazine.Acticoat is made up of nanocrystalline silver particles.Antibiotic-resistant strains have developed rarely to both silvernitrate and silver sulfadiazine but not to nanocrystalline silver. Thebroader range of activity of nanocrystalline silver is apparently due tothe release of both silver cations and uncharged silver species. Due tothe continuing emergence of antibiotic resistant strains of infectiousagents, a need exists for novel antibiotics.

Metal compounds have also played a significant role in other therapeuticapplications. One example of the usefulness of the metals can be seen inthe field of radiopharmaceuticals. The use of radiation therapy todestroy tumor cells is well known, but tumors can reappear aftertherapy. Hypoxic cells within the tumor are 2.5 to 3 times moreresistant to X-ray radiation than other tumor cells. For this reason,these cells are more likely to survive radiation therapy or chemotherapyand lead to the reappearance of the tumor. Targeting of radio nuclidesto hypoxic cells will serve as a method to visualize them.

Complexes of γ-ray emitters such as ⁹⁹Tc are extremely useful as imagingagents, and therapeutic radiopharmaceuticals like ⁸⁹Sr, ¹⁵³Sm, ¹⁸⁶Re and¹⁶⁶Ho are important in the treatment of bone tumors. bRh-105 emits agamma ray of 319 keV (19%) that would allow in vivo tracking anddosimetry calculations. Many more radioactive nuclei can be harnessed byusing the entire periodic table to construct diagnostic or therapeuticagents.

Urinary tract infections (UTIs) represent the second most commoninfectious disease in the United States and are associated withsubstantial morbidity and medical cost. These infections, includingcystitis and pyelonephritis, are most commonly caused by uropathogenicEscherichia coli (UPEC). Patients with neurogenic bladder, indwellingurinary catheters, or vesicoureteral reflux, as well as otherwisehealthy women, experience recurrences; repeated infections of theurinary tract can lead to renal scarring and chronic kidney disease(CKD). Current preventive and therapeutic strategies fail to address theproblem of recurrent UTIs. Recent work in the murine cystitis model hasunveiled new paradigms regarding the pathogenesis of UTI. Long thoughtto be strictly extracellular pathogens, UPEC have been shown to invadesuperficial epithelial cells lining the bladder and to establish largecollections, termed intracellular bacterial communities (IBCs), withinthese cells. From there, UPEC form a quiescent reservoir within bladdertissue that is sequestered from host defenses, resists antibiotictherapies, and can serve as a nidus for recurrence.

The rapid rise in antimicrobial resistance rates among pathogenicstrains renders treatment and prophylactic regimens for UTI increasinglydifficult. For this reason, it is desired to interrogate the utility ofsilver carbenes as novel antimicrobials within the urinary tract. Theantimicrobial properties of silver have been recognized for centuries,and there is recent resurgence of interest in this metal as a biocide.Though silver-impregnated urinary catheters have reduced the incidenceof UTI in certain populations (e.g., patients with indwellingcatheters), novel strategies are needed to prevent recurrent UTI inother patients (e.g., healthy women and patients with functional andanatomic abnormalities of the urinary tract). Organometallic complexesof silver with N-heterocyclic carbenes (NHCs), have been designed andsynthesized. The primary advantage of these silver carbenes (SCs) overexisting silver compounds is their stability and water solubility.

The usefulness of complexes of radioactive metals is highly dependent onthe nature of the chelating ligand. A successful metal drug must bothtarget a specific tissue or organ as well as rapidly clear from othertissues. In addition, for both imaging and tumor treatment, the targetorgan or tissue must have optimal exposure to the radiopharmaceutical.Therefore, there is a need for novel ligand systems designed to bindradioactive metals.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a and 1 b are thermal ellipsoid plots of the cationic portionsof the water soluble silver dimmers shown as Formulas 9a and 9b;

FIG. 2 is a thermal ellipsoid plot of the water soluble diol shown asFormula 13;

FIG. 3 is a thermal ellipsoid plot of the silver carbene complex shownas Formula 17;

FIG. 4 is a thermal ellipsoid plot of the bromide salt shown as Formula20a;

FIG. 5 is a thermal ellipsoid plot of the compound shown as Formula 23;

FIG. 6 is a thermal ellipsoid plot of 5,6,7,8-tetrahydro-5-oxoimidazo[1,5-c]pyrimidine shown as Formula 26;

FIG. 7 is a thermal ellipsoid plot of the compound shown as Formula 27;

FIG. 8 is a thermal ellipsoid plot of the compound shown as Formula 29b;

FIG. 9 is a thermal ellipsoid plot of the compound of the iodide saltshown as Formula 30b;

FIG. 10 is a thermal ellipsoid plot of [PF₆ ⁻] salt of Formula 36;

FIG. 11 is a thermal ellipsoid plot of the silver biscarbene dimmershown as Formula 37;

FIG. 12 is a thermal ellipsoid plot of the tetracationic portion ofFormula 38 [PF₆]₄;

FIG. 13 is a thermal ellipsoid plot of the compound shown as Formula39b;

FIG. 14 is a thermal ellipsoid plot of the tetracationic portion ofFormula 40 [PF₆]₄;

FIG. 15 is a thermal ellipsoid plot of the compound shown as Formula 41;

FIG. 16 is a thermal ellipsoid plot of the dibromide salt show asFormula 43;

FIG. 17 is a thermal ellipsoid plot of the compound shown as Formula 8c;

FIG. 18 is a thermal ellipsoid plot of the compound shown as Formula 8d;

FIG. 19 is a thermal ellipsoid plot of the rhodium carbene shown asFormula 8e;

FIG. 20 is a thermal ellipsoid plot of the compound shown as Formula96b;

FIG. 21 is a thermal ellipsoid plot of the compound shown as Formula97b;

FIG. 22 is a thermal ellipsoid plot of the compound shown as Formula 98;

FIG. 23 is a thermal ellipsoid plot of the compound shown as Formula100;

FIG. 24 is a thermal ellipsoid plot of the salt shown in Formula 108with the thermal ellipsoid drawn at 50% probability level (the counteranions are omitted for clarity);

FIG. 25 is a thermal ellipsoid plot thermal ellipsoid plot of Complex106 with the thermal ellipsoid drawn at 50% probability level (thecounter anions are omitted for clarity);

FIGS. 26 a and 26 b are electrospun fibers prepared from a mixture ofComplex 106 and Tecophilic® at a weight ratio of 25 to 75, where FIG. 26a details as-spun fiber and FIG. 26 b details silver particles formed byexposing the as-spun fiber to water;

FIGS. 27 a and 27 b are TEM images showing the release of silverparticles by exposing fibers of Complex 106 and Tecophilic® (weightratio 50:50) to water vapor environment; FIG. 27 a details as-spun fiberand FIG. 27 b details fibers in water vapor environment for 65 hour;

FIGS. 28 a, 28 b and 28 c are images of the susceptibility test of thefiber mat encapsulating Complex 106, with bactericidal activity comparedto pure Tecophilic® fiber mat, with FIG. 28 a being an image of Complex106/Tecophilic® (weight ratio 25:75), FIG. 28 b being an image of pureTecophilic®, and FIG. 28 c being an image of Complex 106/Tecophilic®(weight ratio 75:25);

FIG. 29 is a graph showing CFU (colony forming unit) versus time (hours)of the silver compounds on S. aureus, expresses the kinetic of thebactericidal activity for each of the silver compounds tested;

FIGS. 30 a, 30 b, 30 c and 30 d are images of electrospun fibers fromComplex 106 and Tecophilic® (weight ratio 75:25) after two weeks ofantimicrobial activity in LB broth media, with FIG. 30 a being a stereoimage of a segment of fiber, FIG. 30 b being an image of large aggregate(400 nm) silver particles encapsulated in Tecophilic® fiber, FIG. 30 cbeing an image of silver aggregates (200 nm to 300 nm in diameter) andsilver particles (10 nm to 20 nm in diameter) in a Tecophilic® matrix,and FIG. 30 d being a top view of a fiber mat with aggregates of silverparticles; and

FIG. 31 is a thermal ellipsoid plot of an N-heterocyclic carbenecompound according to another embodiment of the present invention.

SUMMARY OF THE INVENTION

The present invention generally relates to metal complexes ofN-heterocyclic carbenes that contain one or more additional activemoieties and/or groups therein. In one embodiment, the present inventionrelates to metal complexes of N-heterocyclic carbenes that contain ananti-fungal and/or anti-microbial moiety and/or group in combinationwith one or more additional active moieties and/or groups selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N. In still another embodiment, the present inventionrelates to metal complexes of N-heterocyclic carbenes that contain ananti-fungal and/or anti-microbial moiety and/or group in combinationwith two or more additional active moieties and/or groups selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N.

In one embodiment, the present invention relates to a method for usingand/or administering a silver complex of an N-heterocyclic carbene arerepresented by a compound according to any of the Formulas shown below.In another embodiment, the present invention relates to any silvercomplex of an N-heterocyclic carbene represented by the Formulas shownbelow:

or suitable mixtures of two or more thereof, where R¹, R², R³, R⁴, R⁶and R⁷, if present, are each independently selected from hydrogen;hydroxy; C₁ to C₁₂ alkyl; C₁ to C₁₂ substituted alkyl; C₃ to C₁₂cycloalkyl; C₃ to C₁₂ substituted cycloalkyl; C₂ to C₁₂ alkenyl; C₃ toC₁₂ cycloalkenyl; C₃ to C₁₂ substituted cycloalkenyl; C₂ to C₁₂ alkynyl;C₆ to C₁₂ aryl; C₅ to C₁₂ substituted aryl; C₆ to C₁₂ arylalkyl; C₆ toC₁₂ alkylaryl; C₃ to C₁₂ heterocyclic; C₃ to C₁₂ substitutedheterocyclic; C₁ to C₁₂ alkoxy; C₁ to C₁₂ alcohols; C₁ to C₁₂ carboxy;biphenyl; C₁ to C₆ alkyl biphenyl; C₂ to C₆ alkenyl biphenyl; or C₂ toC₆ alkynyl biphenyl, and where R⁵ is selected from fluoroquinolonecompounds or derivatives thereof; steroids or derivatives thereof;anti-inflammatory compounds or derivatives thereof; anti-fungalcompounds or derivatives thereof; anti-bacterial compounds orderivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N.

DETAILED DESCRIPTION OF THE INVENTION

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionpertains.

The present invention includes a metal complex of a N-heterocycliccarbene, its method of manufacture, and methods of use. Several generaltypes of N-heterocyclic carbene ligands may be used as ligands for ametal such as silver. These include monodentate carbenes, such as thoserepresented by Formula 2, bidentate carbenes such as those representedby Formulas 3 through 5, and bidentate macrocyclic carbenes such asthose represented by Formulas 6 and 7. With the exception of monodentatecarbenes, each of these ligand types has as their basic constituent twoN-heterocyclic carbene units bridged by either methylene groups, as inFormula 3, dimethylpyridine groups, as in Formula 4 and dimethylpyrrolegroups as in Formula 5, or are parts of rings as in Formulas 6 and 7.The water solubility, stability, charge and lipophilicity of silvercomplexes of these N-heterocyclic carbenes may be modified by changes inR₁ and R₂. Each R₁ and R₂, separately or in combination, can be selectedfrom hydrogen, C₁ to C₁₂ alkyl, C₁ to C₁₂ substituted alkyl, C₁ to C₁₂cyclo alkyl, C₁ to C₁₂ substituted cycloalkyl, C₁ to C₁₂ alkenyl, to C₁₂cycloalkenyl, C₁ to C₁₂ substituted cycloalkenyl, C₁ to C₁₂ alkynyl, C₁to C₁₂ aryl, C₁ to C₁₂ substituted aryl, C₁ to C₁₂ arylalkyl, C₁ to C₁₂alkylaryl, C₁ to C₁₂ heterocyclic, C₁ to C₁₂ substituted heterocyclicand C₁ to C₁₂ alkoxy. It is particularly desirable, for at least somepharmaceutical applications, for R₁ and R₂ to be selected such that theresulting metal/N-heterocyclic carbene complex is soluble and stable inan aqueous solution.

In one example, the N-heterocyclic carbene is a bidentate carbenerepresented by Formula 4 or 5, where R₁ is a C₁ to C₆ alkyl or C₁ to C₆hydroxyalkyl group, and R₂ is a hydrogen atom. In one particularexample, the N-heterocyclic carbene is represented by formula 4 or 5,where R₁ is a C₂ to C₃ hydroxyalkyl group, and R₂ is a hydrogen atom. Inanother example, the N-heterocyclic carbene is represented by Formula 4and each adjacent R₁ and R₂ together forms a substituted alkyl group.

As stated above, in one embodiment the present invention also providesnovel N-heterocyclic carbenes represented by the Formula as shown below:

wherein Z is a heterocyclic group, and R₁ and R₂ are, independently orin combination, hydrogen or a C₁ to C₁₂ organic group selected fromalkyl, substituted alkyl, cyclo alkyl, substituted cycloalkyl, alkenyl,cycloalkenyl, substituted cycloalkenyl, alkynyl, aryl, substituted aryl,arylalkyl, alkylaryl, heterocyclic, substituted heterocyclic and alkoxygroups. In one example, Z is a pyridine or a pyrrole. In anotherexample, Z is dimethylpyridine or dimethylpyrrole.

In general, imidazolium salts are the immediate precursors ofN-heterocyclic carbenes. Several procedures may be used to convertimidazolium salts to the corresponding N-heterocyclic carbenes.N-Heterocyclic carbenes may be generated from imidazolium salts bydeprotonation with bases such as KOtBu, KH, and NaH in solvents such asTHF and liquid ammonia. Isolatable N-heterocyclic carbenes may replacetwo-electron donors (such as tetrahydrofuran, carbon monoxide, nitriles,phosphines, and pyridine) on a variety of transition metal complexes togive N-heterocyclic carbene transition metal complexes. However it hasnot always been practical to isolate the carbenes.

N-Heterocyclic carbene complexes may also be obtained by in situgeneration of the N-heterocyclic carbene by deprotonation of thecorresponding imidazolium salts in the presence of a suitable transitionmetal complex. Basic ligands on the metal complex, such as hydride,alkoxide, or acetate can deprotonate the imidazolium salt to form theN-heterocyclic carbene that readily binds to the vacant coordinationsite on a metal. For example Pd(OAc)₂ has been shown to react with avariety of imidazolium salts to form palladium-carbene complexes.

The imidazolium salt can also be treated with an inorganic or organicbase to generate the carbene. The reaction of imidazolium salts withmetals containing basic substituents has been shown to be quite usefulfor the synthesis of transition metal complexes of carbenes. Thecombination of the basic oxide, Ag₂O, with imidazolium salts may be usedto generate silver-carbene complexes. The use of silver-carbenecomplexes as carbene transfer reagents has been used to provide carbenecomplexes of gold(I) and palladium(II). Silver-carbene complexes havebeen employed in this manner to provide complexes with Pd-carbene andCu-carbene bonds. The formation of transition metal-carbene bonds, usingcarbene transfer reagents is favored in many situations because thereactions proceed under mild conditions and without the use of strongbases. For example, the condensation of 2 equivalents of n-butylimidazole or methyl imidazole and 1 equivalent of diiodomethane inrefluxing THF affords the imidazolium salts shown as Formulas 8a or 8bin high yield. The combination of shown as Formulas 8a or 8b with Ag₂Oin water forms the water soluble silver dimers 9a and 9b, respectively.

The combination of two equivalents of 1-iodoethanol (Formula 12) withbisimidazol (Formula 11) in refluxing butanol gives the water solublediol shown as Formula 13. This compound has been characterized by bothNMR and X-ray crystallography.

A similar reaction has been carried out using 1,2-dibromoethane (formula14) with bisimidazol to form the carbene represented by Formula 15. Thealcohol groups of Formula 13 and the bromides of Formula 15 providefunctionalized sites for the incorporation of solubilizing moieties.

The pincer ligands 2,6-bis-(n-butylimidazoliummethyl)pyridine dihalide(Formulas 16a and 16b) are easily obtained by the reaction of N-butylimidazole with 2,6-bis(halogenmethyl)pyridine in a 2:1 molar ratiorespectively. Ligand 16a readily reacts with Ag₂O in CH₂Cl₂ to yield thesilver carbene complex 17. Complex 17 is stable in air and light.

A general synthesis of pincer N-heterocyclic carbenes with a pyridine asthe bridging unit is presented below. The reaction of two equivalents ofpotassium imidazole with 2,6-bis(bromomethyl)pyridine resulted inFormula 19 in 70% yield. The combination of the compound represented byFormula 18 with 2-bromoethanol or 3-bromopropanol gives Formulas 19a and19b, respectively. The combination of the Br salt of Formulas 19a or 19bwith an equimolar amount of Ag₂O gives the silver biscarbene polymers20a and 20b, respectively. Formula 20a has been crystallographicallycharacterized. The bromide salts represented by Formulas 20a and 20b arevery soluble and slowly decompose in water to give a silver mirror onthe side of a flask containing either compound. Formula 20a and itspropanol analog Formula 20b are effective antimicrobials. Derivatives ofthese complexes can be synthesized, using histidine as an exampleprecursor as outlined below, to improve their antimicrobial properties.

The antimicrobial activity of water soluble silver (I) N-heterocycliccarbene 20a, in reference to silver nitrate, was investigated on yeastand fungi (Candida albicans, Aspergillus niger, Mucorales, Saccharomycescerevisiae) using the LB broth dilutions technique, and bacteria (E.coli, S. aureus, P. aeruginosa) of clinical importance. The sensitivitytest of the silver compounds using the Kirby-Bauer agar diffusion(filter paper disk) procedure, shows that silver (I) N-heterocycliccarbenes exhibit antimicrobial activity as effective as silver nitrateon all the bacteria by measuring the zone of growth inhibition usingfilter paper disks impregnated with solutions of the silver compoundplaced on a lawn of organism on an agar plate. Overnight culturescontaining various concentrations of the silver compounds and bacteriaor fungi were examined for growth. For each organism, the tubecontaining the minimum inhibitory concentration (MIC) for each silvercompound was used to inoculate agar plates to confirm the absence ofviable organisms in that culture. Formula 20a was effective on bacteriaand fungi at lower concentrations, and had a longer period of silveractivity than silver nitrate over the 7 day time course of theexperiment. Toxicity studies with rats have shown that ligand 19a, theprecursor to 20a and the material that forms on degradation of 20a, isof low toxicity and clears within two days through the kidneys asdetermined by mass spectroscopy of the urine.

The combination of two equivalents of potassium imidazole (Formula 21)with 2,5-bis(trimethylaminomethyl)pyrrole diiodide (Formula 22) in THFgives Formula 23. Formula 23 has been crystallographically characterizedand its thermal ellipsoid plot is shown as FIG. 5. Addition of twoequivalents of butyl bromide to Formula 23 gives Formula 24 in highyield.

The reaction of histamine dihydrochloride (Formula 25) withcarbonyldiimidazole in DMF resulted in5,6,7,8-tetrahydro-5-oxoimidazo[1,5-c]pyrimidine (Formula 26) in 40%yield. The compound of Formula 26 has been crystallographicallycharacterized (see thermal ellipsoid plot in FIG. 6). The combination oftwo equivalents of Formula 26 with one equivalent of2,6-bis(bromomethyl)pyridine in acetonitrile resulted in the formationof Formula 27 in very high yield.

Methylated histamine and histidine are also expected to have lowtoxicity because histamine and histidine occur naturally in the body.The reaction of L-histidine methyl ester dihydrochloride Formula 28 withcarbonyldiimidazole in DMF results in Formula 29. The combination ofthree equivalents of iodomethane with Formula 29 in refluxingacetonitrile gives Formula 30. The iodide salt of Formula 30 is reactedwith methanol in the presence of N,N-diisopropylethylamine at reflux for3 days to obtain 1-methyl-L-histidine Formula 31. The combination ofthree equivalents of iodomethane with Formula 31 in refluxingacetonitrile gives 1,3-dimethyl-L-histidine Formula 32. The combinationof Formula 32 with Ag₂O in DMSO forms the silver carbene Complex 33.Formula 33b has been shown to have significant antimicrobial activityagainst Staphylococcus aureus, Escherichia coli and Pseudomonasaeruginosa by the Kirby-Bauer technique.

Macrocyclic N-heterocyclic carbenes may be synthesized according to thefollowing method. The reaction of two equivalents of potassium imidazolewith 2,6-bis(bromomethyl)pyridine (Formula 34) resulted in the compoundof Formula 35 in 70% yield. The combination of Formula 35 with thecompound of Formula 34 in DMSO gave the compound of Formula 36 in 80%yield. The combination of the PF₆ ⁻ salt of Formula 36 with an equimolaramount of Ag₂O gives a silver biscarbene dimer (Formula 37) in nearlyquantitative yield. Formulas 36 and 37 have been crystallographicallycharacterized and are represented in FIG. 10 and FIG. 11, respectively.The bromide salt of Formula 37 (X═Br), is soluble and stable in water.Under analogous reaction conditions, the combination of Formula 36 with4 equivalents of Ag₂O gives a tetra-silver biscarbene dimer (not shown,but ref. to as Formula 38 and FIG. 12). The combination of Formula 36(X⁻═Br⁻) with Ag₂O in water directly gives the bromide salt of Formula37. Halide salts of Formula 37 can be synthesized in water, and arewater soluble. The bromide and chloride salts of Formula 37 areeffective antimicrobials.

The 3+1 condensation of the pyrrole shown by Formula 22 (R═H or Me),with the pyridine shown by Formula 18 gives the compound of Formula 39(R═H or Me). Anion exchange of Formula 39a with NH₄ ⁺PF₆ ⁻ gives Formula39b. The combination of Formula 39b (X═PF₆ ⁻, R=Me) with fourequivalents of Ag₂O gives a tetra-silver biscarbene dimer, Formula 40(X═PF₆ ⁻, R=Me), the thermal ellipsoid plot of which is shown in FIG.14.

Addition of one equivalent of Formula 22 to Formula 23 gives thebisimidazolium porphyrinoid of Formula 41 in high yield and on a largescale. Formula 41 has been crystallographically characterized and thethermal ellipsoid plot of the dication ring of Formula 41 is shown asFIG. 15. The combination of Formulas 39 (R═H) and 41 with 4 equivalentsof Ag₂O affords tetra-silver biscarbene dimers analogous to Formulas 38and 40.

The combination of Formula 18 with the bis(bromomethyl)phenanthroline ofFormula 42 affords the expanded macrocycle of Formula 43 as a dibromidesalt.

Monodentate N-heterocyclic carbene silver complexes such as thoserepresented by Formula 48 may be synthesized by the interaction of theimidazolium precursors of Formula 44 with silver oxide. As mentionedabove, the side chains, R, may be chosen so as to modify the watersolubility, lipophilicity and other properties of the complexes. Forexample, R may be hydrogen or a C₁ to C₁₂ organic group selected fromalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, arylalkyl,alkylaryl, heterocyclic, and alkoxy groups and substituted derivativesthereof. Silver complexes such as those represented by Formulas 46 and47, synthesized from histamine and histidine, respectively, can besynthesized and used as antimicrobial compounds. Because histamine andhistidine are present in the body, their derivatives are expected togive the least skin irritation when used as a topical antimicrobial andto provide very limited problems as an internal antimicrobial withexcellent toxicological properties.

The synthesis of the pincer N-heterocyclic carbenes having methene ormethylene groups bridging the two N-heterocyclic carbenes (see Formula3) and with substituents attached is provided below. The substituentsmay be chosen in order to give the overall complex sufficientsolubility, lipophilicity or other properties. Pyridine rings andimidazoles serve as the fundamental building blocks in the proceduresdiscussed below. Based on the synthesis of Formulas 8a and 8b above, twoequivalents of Formula 58 will combine with methylene iodide to form thecompound of Formula 59. Opening of Formula 59 with HCl will provide thecompound of Formula 60. One equivalent of an alkyl halide would readilyadd to the primary amines of Formula 60, because primary amines are morereactive than imidazole nitrogens, to form Formula 61. A second alkylhalide would add to the secondary imidazole nitrogens of Formula 61 toform the bisimidazolium cation shown as in Formula 62. Thebisimidazolium cation 62 may be combined with Ag₂O to form silvercomplexes shown as Formula 63 similar to Formulas 9a and 9b above.

Formula 27 can be treated with HCl to give Formula 64, which can then becontacted with a derivatized alkyl halide containing a solubilizingsubstituent to give Formula 65. Formula 64 could also be derivatizedwith a carboxylic acid and dicyclohexylcarbodiimide (DCC) to form anamide bond. The combination of Formula 65 at a higher temperature with aderivatized alkyl halide that similarly contains a solubilizingsubstituent will give the imidazolium biscation shown as Formula 66,which can be further complexed with metals such as rhodium.

Silver-carbene complexes may also be used as carbene transfer reagentsto create other carbene complexes. The formation of transitionmetal-carbene bonds, using carbene transfer reagents is favored in manysituations because the reactions proceed under mild conditions andwithout the use of strong bases. For example, the combination of Formula8b with Pd(OAc)₂ in DMF followed by treatment with NaI in acetonitrileresults in the formation of the compound represented by Formula 8c. Thethermal ellipsoid plot of this compound is shown below. Similarly, thecombination of Formula 8b with PtCl₂ and sodium acetate in DMSO givesthe compound represented by Formula 8d in 50% yield.

The combination of the imidazolium salt represented by Formula 8a with[(1,5-cyclooctadiene)RhCl]₂ in refluxing MeCN in the presence of NaOAcand Kl gives the rhodium carbene of Formula 8e in 80% yield. Thiscompound has been characterized by ¹H and ¹³C NMR and X-raycrystallography. This rhodium complex is water stable for extendedperiods of time. A related chelating bis-carbene rhodium complex hasbeen synthesized and has been shown to be stable enough to use incatalytic processes.

The silver complex of an N-heterocyclic carbene represented by Formula17 can function as a carbene transfer reagent. The reaction of Formula17 with (PhCN)₂PdCl₂ in CH₂Cl₂ yields the palladium carbene complexrepresented by Formula 67 and two equivalents of AgCl in nearlyquantitative yield.

Similarly, the reaction of the complex represented by Formula 20a with(PhCN)₂PdCl₂ in CH₂Cl₂ yields the palladium carbene complex representedby Formula 68.

A similar synthesis route may be used to synthesize the compoundrepresented by Formula 69 from the compound represented by Formula 19a.

For the synthesis of pyrrole bridged pincer N-heterocyclic carbenes, a2,5-bisdimethylpyrrole with leaving groups on the methyl groups isparticularly useful in the synthesis method of the present invention.The Mannich reaction of dimethylammonium chloride in aqueousformaldehyde and pyrrole gives 2,5-bisdimethylaminomethylpyrrole,represented by Formula 70. Addition of iodomethane to the pyrrolerepresented by Formula 70 in THF gives2,5-bis(trimethylaminomethyl)pyrrole diiodide (Formula 71).

A molecule containing a 2-nitroimidazole group is believed to betargeted to hypoxic cells. These compounds are reduced at thenitroimidazole group and trapped within cells with a low oxygenenvironment. Attachment of a 2-nitroimidazole group to pincerN-heterocyclic carbenes to form the compound represented by Formula 73may be accomplished as follows. The condensation of the compoundrepresented by Formula 72 with bisimidazol in a 2:1 ratio is expected togive the compound represented by Formula 73. Other derivatives of2-nitroimidazole having various linker segments may similarly besynthesized. The variety of linker groups, including polyethylene oxide(PEO), will allow for flexibility in positioning the chelator relativeto the targeting group as well as for variation of the octanol/waterpartition coefficient of the compound, which is relevant to theclearance through the kidneys. The formation of rhodium complexessimilar to Formula 73 is also envisioned. Similar procedures may be usedto synthesize derivatives represented by Formulas 75 and 76 containingnitroimidazole and solubilizing substituents.

Isotopes of the metals indicated herein as components of anN-heterocyclic carbene complex may be used to form radiopharmaceuticals.For example, ¹⁰⁵Rh may be used in place of Rh. ¹⁰⁵Rh has a convenienthalf-life of 1.5 days and also emits relatively low levels ofγ-radiation. This isotope of rhodium decomposes by beta emission to¹⁰⁵Pd a stable naturally occurring isotope of palladium. Otheremployable isotopes can be selected from transition metals, elementsfrom the lanthanide series, and elements from the actinide series.Preferred isotopes are Ag, Rh, Ga, and Tc.

As mentioned above, the present invention includes metal N-heterocycliccarbene complexes that can be made from several N-heterocyclic carbeneprecursors, the imidazolium salts. The imidazolium salts obtained frombiological analogs, such as the purine bases which includes xanthine,hypoxanthine, adenine, guanine and there derivatives can readily bereacted with silver(I) oxide in suitable solvent to obtain thesilver-N-heterocyclic carbene complexes. The imidazolium cations caneasily be classified as mono-imidazolium cation such as thoserepresented by Formulas 77 through 81, bis-imidazolium cations such asthose represented by one of the following Formulas:

Preferable mono-imidazolium cations include those represented byFormulas 48 through 52:

which can be used for the formation of preferred monodentateN-heterocyclic carbene silver complexes, such as those having Formulas53 through 57, respectively. The carbene silver complexes shown inFormulas 53 through 57 can be synthesized by the interaction of theimidazolium precursors 48 through 52, respectively, with a silver oxide:

Similarly, multi-imidazolium cations according to the present inventioninclude those represented by Formulas 82 through 90:

The bis-imidazolium cations bridged can be represented by Z, wherein Zcan be a methylene, heterocyclic group, dimethyl heterocyclic group,dimethyl cycloalkane group, dimethyl substituted heterocyclic group,aryl group, dimethyl substituted aryl group. The bis-imidazolium cationscan be bridge by Z₁ and Z₂ to form a ring (cyclophane), wherein Z₁ andZ₂ can each be separate or in combination, and can be selected fromheterocyclic, C₁ to C₁₂ substituted heterocyclic, aryl, C₁ to C₁₂substituted aryl, C₃ to C₁₂ substituted ketone, and C₁ to C₁₂ alkylenegroups. Each R group; R₁, R₂, R₃ and R₄ functionality, and the counteranion X of the imidazolium salt may be modified to improve thelipophilicity of compound. The X⁻ counter anion may be from halides,carbonate, acetate, phosphate, hexafluorophosphate, tetrafluoroborate,nitrate, methylsulfate, hydroxide and sulfate. Each R group (R₁, R₂, R₃and R₄), separately or in combination, can be selected from hydrogen, C₁to C₁₂ alkyl, C₁ to C₁₂ substituted alkyl, C₁ to C₁₂ alkoxy, C₁ to C₁₂cyclo alkyl, C₁ to C₁₂ substituted C₁ to C₁₂ cyclo alkyl, C₁ to C₁₂alkenyl, C₁ to C₁₂ cycloalkenyl, C₁ to C₁₂ substituted cycloalkenyl, C₁to C₁₂ alkynyl, C₁ to C₁₂ aryl, C₁ to C₁₂ substituted aryl, C₁ to C₁₂arylalkyl, C₁ to C₁₂ alkylamine, C₁ to C₁₂ substituted alkylamine, C₁ toC₁₂ alkylpentose phosphate, C₁ to C₁₂ phenols, and C₁ to C₁₂ esters. Theselection of R₁, R₂, R₃, and R₄ functionality is desirable in some ofits pharmaceutical applications.

Purines are also being examined as carbene precursors for carryingsilver. Of particular interest is guanine, one of the nucleobases inDNA. Guanine 91 has a ring system similar to that of caffeine compoundrepresented by Formula 95. Since guanine is non-toxic it seemsreasonable that 7,9-dimethylguanine would have low toxicity. This makesthe dimethyl guanine ligand very attractive for cystic fibrosis researchbecause we are looking for non-toxic as well as small ligands to serveas carriers for silver cations.

Dimethylation of guanine (see Formula 91) with dimethylsulfate followedby treatment with ammonium hydroxide gives the water insoluble7,9-dimethylguanine zwitterion compound represented by Formula 92.Addition of HBr to the zwitterion compound represented by Formula 92yields the bromide salt of Formula 93. The bromide salt is soluble inwater and is precipitated out using THF. The silver complex is formed bysuspending the bromide salt in DMSO, adding Ag₂O to the solution andheating at 60° C. to 80° C. for about 6 hours.

Xanthines have been used for a number of years as bronchodilators forthe treatment of airway obstructions in cystic fibrosis patients.Because xanthines contain imidazole rings we assumed it should bepossible to alkylate them to form imidazolium cations and eventuallysilver carbene complexes. Because of their use as bronchodilators wealso assumed that their methylated derivatives would be relativelynontoxic. Probably the most well know of the xanthines is the caffeinecompound represented by Formula 95. We have investigated the alkylationof caffeine to form methylated caffeine and the formation of silvercarbene complexes using caffeine as the carbene precursor. Methylatedcaffeine has proven to be even less toxic than caffeine.

The methyl sulfate salt of methylated caffeine,1,3,7,9-tetramethylxanthanium, represented by Formula 96a is produced bythe reaction of the caffeine compound represented by Formula 95 withdimethyl sulfate in nitrobenzene. Anion exchange using NH₄PF₆ in waterresults in the compound represented by Formula 96b.

Ligand 96a is water soluble and reacts with Ag₂O in water to give thecomplex represented by Formula 97a. Formula 97a is stable in water forfive days. The lack of C—¹⁰⁷Ag and C—¹⁰⁹Ag couplings suggests fluxionalbehavior on the ¹³C NMR timescale as observed with many silver(I)complexes. Similarly, Formula 96b reacts with Ag₂O in DMSO to form thecompound represented by Formula 97b, which has been structurallycharacterized by X-ray crystallography. The thermal ellipsoid plots(TEP) of the cationic portions of Formulas 96b and 97b are shown below.

Caffeine, 1,3,7-trimethylxanthine, is one of the xanthine derivativesthat are generally used in medicines as diuretics, central nervoussystem stimulants and inhibitors of cyclic adenosine monophosphate(c-AMP) phosphodiesterase. 1,3,7,9-tetramethylxanthinium iodide(methylated caffeine), an imidazolium salt, was synthesized usingmodified literature procedures and characterized by ¹H, ¹³C NMR, massspectrometry and X-ray crystallography.

The reaction of two equivalent of 1,3,7,9-tetramethylxanthinium iodidewith three equivalent of silver(I) oxide in methanol at room temperatureyields the compound represented by Formula 99.

The crystallization of the compound represented by Formula 99 in amixture of methanol and ethyl acetate yields the compound represented byFormula 100, a colorless crystal, soluble in water and air stable. Thecompounds represented by Formulas 99 and 100 were characterized by ¹H,¹³C NMR, and mass spectrometry. X-ray crystallography was used toconfirm the molecular structure of the compound represented by Formula100 with the thermal ellipsoid plot show above. The antimicrobialproperties of the compound represented by Formula 100 have beenevaluated using both the filter disk test and the standard MICtechnique. The compound represented by Formula 100 was found to haveeffective antimicrobial activity on S. aureus, P. aeruginosa, and E.coli. The dose-response effect on the compound represented by Formula 98was assessed to determine the toxicity of the compound on rats. Thetoxicity study, is a standard protocol used to determine the lethal doserequired to kill half (LD 50) of the animals (rats). The LD 50assessment on the compound represented by Formula 98 was 2.37 grams perkilogram of rat. The protocol used in this study was approved by theInstitutional Animal Care and Use Committee (IACUC), University ofAkron.

The delivery methods for administering an effective amount of transitionmetal complexes of N-heterocyclic carbenes for in-vitro and in-vivomedicinal application consist of aerosol, biodegradable polymers,polymeric micelles, hydrogel types materials, dendrimers, and modifiedC-60 fullerenes.

The reaction shown resulting in the silver carbene complex representedby Formula 202 is similar in nature to the silver carbene complexrepresented by Formula 100. The compound represented by Formula 202 isan additional silver complex of xanthine derivative, namely7-(2,3-dihydroxypropyl)theophylline silver(I) complex. The compoundrepresented by Formula 202 is a derivative of theophylline complexedwith Ag (I) that has a K_(sp) of 82 mg/mL (attributable to the hydroxylgroup), and is stable in solid form for periods of a couple months.Synthesis of the compound represented by Formula 202 is adaptable tolarge scale production.

The imidazolium salt 1,3,9-trimethyl-7-(2,3-dihydroxypropyl)xantheniumiodide represented by Formula 201 is obtained by reacting7-(2,3-dihydroxypropyl)theophylline (Formula 200) with methyl iodide indimethylformamide. The imidazolium salt represented by Formula 201reacts with silver acetate in methanol to yield the N-heterocycliccarbene silver(I) acetate complex (Formula 202), which is a white solidin 34% yield (structure confirmed by X-ray crystallography). Thecompound represented by Formula 202 is water soluble (K_(sp)=82 mg/mL)and is stable for at least 7 days in water by NMR. As the compoundrepresented by Formula 202 decomposes to release Ag⁺ therebyregenerating the cationic portion of the compound of Formula 201. Theimidazolium cation portion of the compound represented by Formula 201has an LD₅₀ in rats of >2.0 g/kg in preliminary studies.

The compound represented by Formula 202 has a shelf life of severalmonths at room temperature. Each portion of the compound represented byFormula 202 can be readily reconstituted in sterile water to form aclear, colorless solution with a concentration of 10 mg/mL.

In addition to the imidazole ring portion that is converted into acarbene for binding metals, the feature common to the compoundsrepresented by Formulas 100, 202 and the generic form of 56 is thepresence of a bis-amide ring on the “backside” of the imidazole-carbeneportion. This bis-amide ring is electron withdrawing. Silver acetatecarbene complexes that do not contain electron-withdrawing groups in thering are not as stable in water as the compounds represented by Formulas100 and 202.

The minimum inhibitory concentrations (MIC) of the silver carbenecompound represented by Formula 202 for a panel of E-coli from a varietyof sources was determined (Escherichia coli being the leading cause ofurinary tract infections). Strains influenced included the sequencedcystitis strain UTI89 and pyelonephritis strain CFT073; the sequencedlaboratory E. coli strain MG1655; and seven strains from patients withacute or recurrent UTIs or asymptomatic bacteriuria. Overnight Luriabroth (LB) cultures of these strains were sub-cultured 1:100, grown 2 to3 hours to OD_(600nm)=0.4, and diluted 1000-fold in fresh LB. Onehundred μL of each suspension was added to 100 μL of a range ofdilutions of the compound represented by Formula 202 in wells of a96-well plate. After 16 hours static incubation at 37° C., MICs wereassessed visually and by quantitative absorbance measurement in amicroplate reader at 600 nm. The MIC of the compound represented byFormula 202 against this panel of strains was generally 2 to 4 μg/mL,similar to that observed against P. aeruginosa and Burkholderia species.

A prerequisite for a topical biocide is that it confers acceptabletoxicity to the tissue(s) of interest. In vitro toxicity of the compoundrepresented by Formula 202 has been studied using the bladdercarcinoma-derived T24 epithelial cell line (ATCC HTB-4). T24 cells weregrown in RPMI 1640 medium available from Life Technologies (Carlsbad,Calif.) supplemented with 10% fetal bovine serum available from Sigma(St. Louis, Mo.), seeded into 24-well plates, and grown to confluenceover 48 hours. Cells were washed with sterile phosphate buffered saline(PBS) and fresh warmed medium was added, either alone or containing thecompound represented by Formula 202 (added to the medium at the start ofthe experiment to minimize premature liberation of Ag⁺ from the compoundrepresented by Formula 202) at concentrations between 5 and 50 μg/mL.After incubation for 1 to 2 hours, cells were released by treatment with0.05% trypsin −0.02% EDTA, suspended in sorting buffer, stained withpropidium iodide, and subjected to flow cytometry on a FACS Caliburinstrument available from Becton Dickinson (Piscataway, N.J.). Ourinitial experiments demonstrate that loss of viability is ˜5% after 1 hof treatment with 202 at 5 μg/mL and approximately 11% after 2 hours ofsuch treatment.

Additional work has explored the addition of electron-withdrawing groupson the “backside” of the carbene moiety, which provides augmentedstability to sodium, chloride, and other ions. Deprotonation of thecompound represented by Formula 205 with potassium hydroxide followed bydouble methylation with methyl iodide gives the N-heterocyclic carbenecompound represented by Formula 206. The bis(NHC) silver(I) complexrepresented by Formula 208 was formed from the reaction of the nitratesalt compound represented by Formula 207 with an excess of silver(I)oxide in acetonitrile.

The bis(NHC) silver(I) complex, Formula 210, with an iodide anion wasadded to 0.9% sodium chloride solution (equivalent to physiologicalserum Na⁺ concentration). The solution was decanted and the resultingprecipitate was dissolved in acetone. Slow evaporation of acetone yieldwhite crystals of a compound represented by Formula 211 that showbridging chlorides and a silver NHC bond still intact. The stability ofthe compound represented by Formula 211 to physiological concentrationsof sodium chloride is unprecedented. In addition to stabilizing silverNHCs to water, the presence of electron-withdrawing groups on theimidazole ring can greatly enhance their stability to physiologicalsodium chloride. This type chemistry is particularly suited for use inthe urinary tract, where urinary osmolality in humans may vary from 300to 1200 mOsm/L.

As stated above, the major advantage of SCs over earlier silvercompounds is their stability and solubility in water. The addition ofelectron-withdrawing groups on the “backside” of the carbene componentprovides augmented stability to ionic strength, such as might be foundin the urinary tract.

The 4,5-dihaloimidazoles, Formula 215, were also explored for theirability to form stable silver NHC complexes. The imidazolium saltrepresented by Formula 216 is synthesized using the appropriatemethylating agents. Imidazole starting materials with otherelectron-withdrawing groups such as nitro and cyano groups, Formula 217,are examined. The dinitro and dicyano analogs of the compoundrepresented by Formula 217 are commercially available and synthesis ofthe cyano-nifro analog is known. The imidazolium salts of thesecompounds, Formula 218, are synthesized according to the generalprocedure outlined as before. The compounds represented by Formulas 216and 218 are then be combined with silver acetate and silver oxide toform new silver carbene using procedures discussed above.

The delivery methods for administering an effective amount of silvercomplexes of N-heterocyclic carbenes for in-vitro and in-vivo medicinalapplication consist of (or include) aerosol, biodegradable polymers,polymeric micelles, hydrogel types materials, dendrimers, and modifiedC-60 fullerenes. The silver carbene complexes are used in an amount from0.01 μg to 600 mg. The preferred delivery method for treating urinarytract infections using silver carbene complexes involves dissolving thesilver carbene complex into a fluid such as, but not limited to, wateror saline. Water and saline are preferred due to their compatibilitywith the human body, but other fluids can be used as well depending uponthe application. The silver carbene complex solution is instilled intothe urinary bladder via an instrument such as, but not limited to, aurinary catheter. A normal sized urinary bladder in an adult human is500 to 600 mL. The preferred amount of fluid used in treatment ofurinary tract infections is 1 to 600 mL, another preferred range is 25to 450 mL, and another preferred range is 80 to 300 mL. The preferredconcentration of the silver carbene complex in fluid is in the range of0.01 to 1000 μg/mL, another preferred range is 0.5 to 100 μg/mL andanother preferred range is 1 to 25 μg/mL.

Regarding urinary tract infections the terms treating and/or treatmentinclude resolving an existing urinary tract infection and/orpre-treating a bladder to prevent the initiation of a urinary tractinfection. Such pretreatments would benefit patients at-risk for urinarytract infections. Pre-treating the bladder for a urinary tract infectioninvolves the same method of filling the bladder as treatment, but withless frequency. For example, patients practicing clean intermittentcatheridization would adhere to a periodic schedule such as but notlimited to once a month, once a week or once a day.

In order to demonstrate the practice of the present invention, twoN-heterocyclic carbenes represented by Formulas 101 and 102 weresynthesized and tested for antimicrobial properties as described below.The compounds can be shown with reference to Formula 4:

where R₁ is a hydroxyethyl or hydroxypropyl group and R₂ is a hydrogenatom. These carbenes 101 and 102 were synthesized by reacting2,6-bis-(imidazolmethyl)pyridine with either 2-iodoethanol or3-bromopropanol to provide compounds of Formulas 101 and 102.

The IR spectra for these compounds show an O—H stretching bandvibration, 3325 cm⁻¹. FAB-MS spectra obtained from these compounds innitrobenzyl matrices showed [51][I]⁺ (C₁₇H₂₃N₅O₂I) at m/z 456 and[52][I]⁺ (C₁₉H₂₇N₅O₂Br) at m/z 436. These compounds readily react withAg₂O to form the silver-bis(carbene) pincer complexes represented byFormulas 103 and 104 in high yield.

The formation of the compounds represented by Formulas 103 and 104 isconfirmed by the loss of the imidazolium proton at 9.13 ppm, 9.36 ppm inthe ¹H NMR spectra of these compounds, and the appearance of a resonanceat 181 ppm in the ¹³C NMR spectra of these compounds. Further evidencefor the formation and structure of compound 103 is provided by X-raycrystallography.

Colorless crystals of the compound represented by Formula 103 wereobtained by slow evaporation of a methanol solution of the compoundrepresented by Formula 103. Interestingly, the compound represented byFormula 103 undergoes complete anion exchange in aqueous methanol,replacing the iodide anions with hydroxide anions. In the solid state,the compound represented by Formula 103 exists as a one-dimensionallinear polymer as shown in FIG. 1. FIG. 1 is a thermal ellipsoid plot ofthe compound represented by Formula 103 with the thermal ellipsoid drawnat a 30 percent probability level. The hydrogen atoms have been omittedfrom FIG. 1 for clarity.

The geometry at the silver atoms is nearly linear with a C5-Ag1-C15 bondangle of 174.7(4)°, and Ag1-C15, and Ag1-C15 bond distances of 2.108(11)Angstroms and 2.060(13) Angstroms, respectively. Mass spectroscopysuggests that in solution and in the gas phase, the compound representedby Formula 103 exists as monomer, whereas X-ray crystallography showsthat the compound represented by Formula 103 is polymeric in thecrystal.

An anion exchange reaction of the compound represented by Formula 103with aqueous ammonium hexafluorophosphate results in the formation ofthe compound represented by Formula 105. In the solid state, thecompound represented by Formula 105 exists as a dimer, as shown in FIG.2. FIG. 2 is a thermal ellipsoid plot of the compound represented byFormula 105 with the thermal ellipsoid drawn at a 30 percent probabilitylevel. The hydrogen atoms have been omitted from FIG. 2 for clarity. Thegeometry of the silver atoms are nearly linear withC32-Ag1-05)(175.7(4)°, C22-Ag2-C17) (174.6(3)° bonds angles, and Ag1-C32(2.070(9) Angstroms), Ag1-05 (2.091(9) Angstroms), Ag2-C22 (2.064(9)Angstroms), Ag2-C17 (2.074(8) Angstroms) bond lengths. The nature of theanions is significant to the structural changes of the compoundrepresented by Formula 103 versus the compound represented by Formula105, and the choice of anion has a pronounced effect on the solubilityof these compounds. For example, the compound represented by Formula 103is soluble in aqueous media whereas the compound represented by Formula105 is not. Table 1 gives a summary of the crystal data of both of thesecompounds.

TABLE 1 Empirical Formula 103, C₁₇H₂₂N₅O₃Ag 105, C₃₄H₄₂N₁₀O₄AgP₂F₁₂Formula Weight 434.0735 868.1481 Temperature (K) 100 100 Wavelength (Å)0.71073 0.71073 Crystal system, Orthorhombic, Monoclinic, P2(1)/c, 8space group, Z P2(1)2(1)2(1), 4 Unit cell dimensions a (Å)  4.5586(17)10.9448(14) b (Å) 14.900(6) 22.885(3) c (Å)  29.923(12) 17.729(2) α (°)90 90 β (°) 90 92.196(2) γ (°) 90 90 V (Å3)  2032.5(14)  4437.4(10)Dcalc (Mg/m3) 1.422 1.737 Absorption 1.010 1.055 coefficient (mm-1)Theta range for 1.36 to 24.99 1.45 to 25.00 data collection (°)Reflections 6300/3506 20811/7757 collected/unique [R(int) = 0.0650][R(int) = 0.0437] Goodness-of-fit 1.034 1.058 on F2 Final R indices0.0655 0.0956 [I > 2 σ (I)] R indices (all data) 0.1410 0.2491 Largestdifference 0.954 and −0.875 2.069 and −1.230 peak and hole (e Å−3)

The usefulness of the compounds represented by Formulas 103 and 55 asantimicrobial agents was evaluated. The standard agar plates overlaymethod was used to obtain the sensitivity data as presented in Table 2.In this test, a filter paper disc of 6 mm diameter was soaked with 20 μLof a silver compound of known concentration, and placed over a lawn ofan organism in the agar plate. The diameter of the area in which growthof the organism is inhibited by the test solution was measured after anover night incubation as a measure of the relative antimicrobialactivity of the silver compounds. The test organisms were Escherichiacoli, Staphylococcus aureus, and Pseudomonas aeruginosa. Silver nitratewas the reference standard used, while the compounds represented byFormulas 101 and 102 served as a negative controls.

TABLE 2 Antimicrobial Activity of Silver Compounds Ag⁺ E. coli S. aureusP. aeruginosa Tested compounds (μg/mL) Diameter of Inhibited Area (mm)AgNO₃ - 0.5% (w/v) 3176 11.38 10.88 11 Compound 103 - 1.31% 3130 11.5 1112 Compound 105 - 1.42% 3195 11.58 10.67 10.25 Compound 103 - 0.50% 119510.13 10 11.13 Compound 105 - 0.50% 1125 10 9 12 Compound 101 - 0.50% 66 6 Compound 102 - 0.50% 6 6 6

The data confirmed that compounds 103 and 105 have antimicrobialproperties at a level comparable to silver nitrate as shown in Table 2.The pincer ligands, compounds 101 and 102, were found to have noantimicrobial activity.

The silver compounds were also tested according to the minimuminhibition concentration determination method (MIC). The MIC is astandard microbiological technique used to evaluate the bacteriostaticactivity of antimicrobial agents. In this case, the MIC was based on thetotal amount of silver available and not on the concentration of silverions. A 0.5 percent (w/v) solution of each of the silver compounds 103and 105 was tested. On dissolving of the silver complexes in the culturemedium (LB broth), a precipitate of AgCl was observed in all samples.The activity of a dilution series of the supernatant portion of thesilver complex solutions was evaluated, with the addition of a constantvolume of freshly grown organism (20 μL) per day. Escherichia coli,Staphylococcus aureus, and Pseudomonas aeruginosa were again used as thetest organisms. The MIC was obtained by visual inspection of thecultures for growth(+) or no growth(−) as reported in Table 3. In Table3, DF is the dilution factor. From the results, it can be concluded thatcompounds 103 and 105 are less bound to chloride ion than silvernitrate, due to the stability of the Ag—C donor ligand bond. Thus,compounds 103 and 105 show better antimicrobial activity than silvernitrate. This is a desirable property of compounds 103 and 105, whenconsidering silver compounds for in vivo application. It may be notedthat although equal weights of silver compounds were used, the amount ofsilver ions released by compounds 103 and 105 is about 2.7 times lowerthan the amount of silver ions released by silver nitrate.

TABLE 3 MIC Results of Supernatants of Silver Compounds (less silverchloride) Test Ag Ag E. coli P. aeruginosa S. aureus compounds (ul/ml)Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 103 1186 − − − − − − ×1DF − + − −− + ×2DF − + − + + ×3DF + + + ×4DF + + + 105 1125 − − − − − − ×1DF − +− + − + ×2DF − + − + + ×3DF + + + ×4DF + + + AgN03 3176 − + − + +×1DF + + + ×2DF + + + ×3DF + + + ×4DF + + +

While not wishing to condition patentability on any particular theory,it is believed that the activity and stability of compounds 103 and 105,as well as their solubility in water, may be attributed to therelatively slow decomposition of Ag—C donor ligand bond over time tosilver metal and silver ion.

When the MIC test was repeated as described above except in the presenceof insoluble silver chloride, the activity of the silver compounds wasenhanced, with silver nitrate performing better as shown in Table 4. Ithas been previously reported that the presence of chloride contributesto the toxicity of silver in sensitive strains of organisms.

TABLE 4 Effect of Chloride (as Silver Chloride) in the BactericidalActivity of Silver Compounds Tested Ag E. coli P. aeruginosa S. aureuscompounds (Days) (Days) (Days) (% w/v) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 56 103 0.50 − − − − − − − − − − − − − − − − − − 0.25 − − − − − − − − − −− − − − − − − − 0.12 − − − − − − − − − − − − − − − − − − 0.06 − − − − −− − − − − − − − − − − − − 0.03 − − + − − + − + 105 0.50 − − − − − − − −− − − − − − − − − − 0.25 − − − − − − − − − − − − − − − − − − 0.12 − − −− − − − − − − − − − − − − − − 0.06 − − − − − − − − − − − − − − − − − −0.03 − − + − − + − + AgNO₃ 0.50 − − − − − − − − − − − − − − − − − − 0.25− − − − − − − − − − − − − − − − − − 0.12 − − − − − − − − − − − − − − − −− − 0.06 − − − − − − − − − − − − − − − − − − 0.03 − − − + − − + − − +

The minimum lethal concentration was determined to evaluate thebactericidal properties of the compounds represented by Formulas 103 and105. The clear (no growth) portion of the culture media with the lowestAg compound concentration was used, by streaking 0.01 mL of the solutionon agar plate using a sterilized loop followed by incubation at 37° C.for 24 to 48 hours. The colonies were visually counted, with the endpoint of the minimum bactericidal concentration (MBC) as no growth onthe agar plate. The test compounds showed an improved bactericidaleffect compared to silver nitrate up to the seventh day of incubationand MBC test, with no growth observed after the tenth day of incubationand testing for the silver compounds. This is despite the fact thatfreshly grown organisms were added each day to the culture mediacontaining the silver compounds throughout the incubation period. Thebactericidal and bacteriostatic properties of 103 and 105 are believedto be due to the slow decomposition of the Ag—C donor (carbene) ligandbond over time to silver metal, silver ion, AgCl and to their solubilityin water.

The alkanol N-functionalized silver carbene complexes represented byFormulas 103 and 105 are soluble in aqueous media. In addition, theyhave proved to be useful antimicrobial agents, and their solubility inwater makes them excellent silver compounds that can be of use for invivo application. The solubility and stability of silver complexes inchloride solution have been key factors that have limited the use ofsilver complexes for in vivo application.

According to another aspect of the present invention, a silver(I)imidazole cyclophane gem diol complex 106 [Ag₂C₃₆N₁₀O₄]²⁺ 2(x)⁻, wherex=OH⁻, CO₃ ²⁻ was synthesized. The MIC test showed that theantimicrobial activity of the aqueous form of 106 is 2 fold lesseffective than 0.5% AgNO₃, with about the same amount of silver. Theantimicrobial activity of 106 was enhanced when encapsulated intoTecophilic® polymer by electrospinning (technique) to obtain mats madeof nano-fibers. The fiber mats release aggregates of silvernanoparticles and sustained the antimicrobial activity of the mats overa long period of time. The rate of bactericidal activity of the compoundrepresented by Formula 106 was greatly improved by encapsulation, andthe amount of silver used was much reduced. The fiber mat of thecompound represented by Formula 106 with 75% (106/tecophilic) contained2 mg of Ag, which is 8 times lower than 16 mg (0.5%) AgNO₃ and 5 timeslower than silver sulfadiazine cream 1% (10 mg). The fiber mat was foundto kill S. aureus at the same rate as 0.5% AgNO₃, with zero colonies onan agar plate and about 6 hours faster than silver sulfadiazine cream.Inoculums tested on and found effective are E. coli, P. aeruginosa, S.aureus, C. albicans, A. niger and S. cerevisiae. Transmission electronmicroscopy and scanning electron microscopy were used to characterizethe fiber mats. The acute toxicity of the ligand (imidazolium cyclophanegem diol dichloride) was assessed by intravenous administration to rats,with an LD 50 of 100 mg/Kg of rat.

An electrospun fiber of the present invention can encapsulate asilver(I) N-heterocyclic carbene complex. The antimicrobial activity ofsilver(I)-N-pincer 2,6-bis(hydroxylethylimidazolemethyl)pyridinehydroxide, a water soluble silver(I) carbene complex 107, on someclinically important bacteria was described above. Compound 107 is anexample of a compound that is sparingly soluble in absolute ethanol butcompletely soluble in methanol. The solubility of type 1 silver(I)carbene complexes in ethanol, was improved by varying the functionalizedgroups coupled to the nucleophilic end of thebis(imidazolmethyl)pyridine compound. Although embodiments wherein m=2and m=3 are shown in Formula 107, m can have any positive integer valuethat is at least 1, and preferably, m has a value within the range ofabout 1 to about 4. Further, alternate starting materials or precursorsdescribed above may be used to produce a desired silver(I) carbenecomplex without departing from the scope of the present invention. Thespecific embodiments illustrated and described below are used forillustrative purposes in describing the present invention.

where (a) m=2 and (b) m=3.

Electrospinning is a versatile method used to produce fibers withdiameters ranging from a few nanometers to over microns by creating anelectrically charged jet of polymer solution or polymer melt, whichelongates and solidifies. The resulting fibers can be used in filters,coating templates, protective clothing, biomedical applications, wounddressing, drug delivery, solar sails, solar cells, catalyst carriers,and reinforcing agents for composites.

The imidazolium (NHC) cyclophane gem-diol salt 108 can be prepared byreacting 2,6-bis(imidazolmethyl)pyridine with 1,3-dichloroacetone asshown below in Equation 2. The formation of the salt compoundrepresented by Formula 108 as a gem-diol in preference to the carbonylform is not expected with electron withdrawing groups present. Withoutbeing bound to theory, it is believed that the formation of the saltcompound represented by Formula 108 as a gem-diol proceeded byacid-catalyzed process with the solution observed to be slightly acidichaving a pH range of 5 to 6.

The ¹H NMR spectra showed the presence of gem O—H as a broad peak at7.65 ppm, and the absence of C═O in the salt compound represented byFormula 108 was observed in both ¹³C NMR and IR spectroscopy. The O—Hstretching vibration was observed at 3387 cm⁻¹, while the C—O stretchingat 1171 cm⁻¹ and ¹³C NMR chemical shift at 91 ppm. The x-raycrystallography further provided the evidence and structure of thecompound represented by Formula 108 as shown in FIG. 24.

The combination of silver(I) oxide with the salt compound represented byFormula 108 in methanol according to the reaction scheme illustrated inEquation 3 results in the complex represented by Formula 106 as an airand light stable yellow solid in high yield, confirmed by the loss ofthe imidazolium proton at 9.35 ppm of the ¹H NMR spectra. The proton NMRof the complex compound represented by Formula 106 showed a broad signalwith complicated peaks that are not easily assigned. Again, withoutbeing bound to theory, this may be due to the fluxional behavior of thecompound on the NMR time scale.

The shift in the resonance signal of the imidazole carbon (NCN) from 138ppm to downfield of the ¹³C NMR spectra at 184 and 186 ppm shows therare coupling of the Ag—C bond. The large value of the Ag—C couplingconstant (J_(Agc)=211 Hz) observed agreed with the reported range of 204Hz to 220 Hz for ¹⁰⁹Ag nuclei coupling. ¹⁰⁹Ag coupling is commonlyobserved due to its higher sensitivity compared to the ¹⁰⁷Ag. The x-raycrystallography confirms the structure of complex 106, which is shown inFIG. 25, with bond distances of Ag1-C15=2.085(5) Angstrom,Ag1-C31=2.077(5) Angstrom, Ag2-05=2.073(5) Angstrom and Ag2-C21=2.072Angstrom. A weak Ag1-Ag2 interaction was observed with a bond length of3.3751(10) Angstrom, longer than the commonly reported Ag—Ag bond rangeof 2.853-3.290 Angstrom, but shorter than the Van der waals radii forAg—Ag of 3.44 Angstroms. In silver metal the Ag—Ag bond distance isknown to be 2.888 Angstroms. The C—Ag—C bond angles are almost linearwith C15-Ag1-C31 bond angle of 175.20(18)° and C21-Ag2-05 bond angle of170.56(18)°.

The electrospun fibers from Tecophilic® and silver complex werecharacterized by transmission electron microscopy (TEM) and scanningelectron microscopy (SEM). No obvious phase separation was observed inas-spun fibers, shown in FIG. 26, which indicated a generally-uniformmixing of Tecophilic® and silver complex. The thickness of the fiber matwas measured by scanning electron microscopy (SEM) with pureTecophilic®(100 micron), 25:75 silver complex 106/Tecophilic® (30microns) and 75:25 complex 106/Tecophilic® (60 microns) respectively.The encapsulation of complex 106 by polymer retards the quickdecomposition of silver complex into silver ions or particles in anaqueous media. The formation of silver particles at nanometer scale hasbeen observed in the polymer matrix, when the electrospun fiber isexposed to water. Transmission electron microscopy studies showed thatthe activation of nanosilver particles in the fiber is a process thatoccurs gradually over a period of time. By exposing the as-spun fibersto water, complex 106 decomposed and release silver ions whichaggregated into silver particles at nano-scale measurement. Theformation of aggregates of silver particles has been observed within 30minutes of exposure to water vapor (as shown in FIG. 27). Theaggregation of the silver ions in the presence of water, with theaggregate adsorbed on the surface of the fibers is considered to be asimplified mechanism by which the fiber mat releases the active form(s)of the silver for its antimicrobial activity. The fiber of complex 106is stable in light and air for months, but sensitive to an environmentwith very high humidity.

Bactericidal Effect:

Using a modified Kirby Bauer technique mats of electrospun Tecophilic®fiber encapsulating complex 106 and pure electrospun Tecophilic® fiberas control were placed on a lawn of organism in an agar plate andincubated overnight at 35° C. The inocula used were both Gram positiveand Gram negative prokaryotes (Escherichia coli, Pseudomonas aeruginosa,and Staphylococcus aureus) of clinical interest. The fungi used wereCandida albicans, Aspergillus niger, and Saccharomyces cerevisiae. Thebactericidal activity showed a clear zone of inhibition within andaround the fiber mat after an overnight incubation of the agar plate at35° C. The fungicidal activity was observed after 48 hrs of incubationat 25° C. Pure Tecophilic® fiber mat as control showed no growthinhibition (see FIG. 28). No obvious difference was observed in thediameter of the cleared zone of inhibition around the fiber mat when thecomposition of the fiber mat was changed from 75% of complex 106 and 25%Tecophilic® to 25% of complex 106 and 75% Tecophilic®. The diameter ofthe zone of inhibition for the 75% (complex 106/Tecophilc®) fiber mat is4.00 mm while that of 25% (complex 106/Tecophilc®) is 2.00 mm. Thedifference in diameter of the zone of inhibition between the two typesof fiber mat has no linear relationship with the amount of silver (3:1ratio) present in the two fiber mats. These result further shows thelimitation of the Kirby Bauer technique as a quantitative tool todetermine the antimicrobial activity of drugs. The diffusing ability ofthe silver ions might have been limited by the formation of secondarysilver compounds. Ionic silver is known to undergo ligand exchangereactions with biological ligands such as nucleic acids, proteins, andcell membranes.

Deposition of a few silver particles was observed at the bottom of atest tube when a piece of the fiber mat was placed in 5 mL of distilledwater and exposed to light for 4 days. The leaching of the silverparticles from the fiber mat surfaces to the solution occurred graduallyover time. The release of nano-silver particles from the as-spun mats ofcomplex 106 into an aqueous medium lead to the investigation of thekinetics of kill (bactericidal activity) of the as-spun fiber mat ofcomplex 106 with respect to time by comparing it with silver nitrate andsilver sulfadiazine 1% cream or silvadene (SSD), a clinical drug widelyin use. Both types of the fiber mat composition 75:25 (amount of Ag=424μg/mL) and 25:75 (amount of Ag=140 μg/mL) used in this study showed afaster kill rate than SSD (amount of Ag=3020 μg/mL). Silver nitrate(0.5%) with 3176 μg/mL of Ag showed about the same kill rate as complex106/tecophilic 75:25 (Ag=424 μg/mL) at a silver concentration 8 foldlower than silver nitrate (see FIG. 29). Bactericidal activity of thesilver compounds is faster on P. aeruginosa than on S. aureus. The fibermats killed bacteria faster and better than silvadene.

The time dependence of the bacteriostatic and bactericidal activities ofthe as-spun mat of complex 106 as a function of the volume of organisminoculated was examined. The fiber mats of complex 106 showed aneffective bactericidal activity on P. aeruginosa, E. coli and S. aureusfor over a week with daily inoculation (25 μL) of freshly grownorganism.

This is an indication that the as-spun fiber mat sustained thecontinuous release of active silver species over a long period of time.Pure Tecophilic® mat as control showed no antimicrobial activity within24 hours of incubation. The as-spun mat of complex 106 with the 75%complex 106/tecophilic composition showed better bactericidal effect onP. aeruginosa than the 25% complex 106/tecophilic for over 2 weeks afterinoculating with over 200 μL (2×10⁷) of freshly grown organism.Bacteriostatic activity was observed for S. aureus and E. coli after 10days of the daily streaking of the LB broth solution on an agar plate.Visual inspection of the incubated solutions showed no growth of theorganism.

The bactericidal activity of 108, complex 106 and AgNO₃ in aqueous LBbroth was studied using the minimum inhibitory concentration (MIC) test.There was generally no difference in the bactericidal activity and MICof complex 106 and AgNO₃ after 24 hours of incubation as shown in Table5. However, after 48 hrs of incubation, silver nitrate showed a betterantimicrobial activity at a concentration 2 fold lower than complex 106(838 μg/mL).

TABLE 5 MIC Result Comparing the Activity of AgNO₃ and Complex 106, withboth having about the Same Amount of Silver Conc. of Conc. of Vol. of E.coli P. aereginousa S. aureus Sample sample sample bacteria (Day) (Day)(Day) ID (wt/V %) (μg/mL) (μL) 1 2 1 2 1 2 AgNO₃ 0.50 3462.35 100 − − −− − − 1DF 1731.18 − − − − − − 2DF 865.59 − − − − − − 3DF 432.79 − − − −− − 4DF 216.40 − + − − − + 106 1.38 3341.48 100 − − − − − − 1DF 1675.74− − − − − − 2DF 837.87 − − − − − − 3DF 418.94 − + − + − + 4DF 209.47 − +− + − + 108 0.5  25 + + + MIC result comparing the activity of AgNO₃ and106, with both having about the same amount of silver. DF is thedilution factor (1 mL); + = growth; − = no growth. The amount of silver(μg) per mL for each compound was calculated as (molecular mass ofAg/formula wt of compound) × wt %.

The MIC value was not determined for silver sulfadiazine because of thecloudy nature of the solution, and the concentration of 108 used showedno antimicrobial activity. The dilutions with the least concentration ofcomplex 106 (209 μg/mL) and AgNO₃ (216 μg/mL) in the MIC test wasobserved to show growth of the same number of colonies of S. aureus onan agar plate after 24 hours of incubation. The 25% complex106/tecophilic fiber mat has the least concentration of Ag, 140 μg/mL(see Table 6), and sustain the release of active silver species thatwere bio-available for days. No growth of the organism was observed withthe daily increase in the volume of inocula.

TABLE 6 Showing Details of Silver Compounds used for the Kinetic StudiesWt of Ag Volume compds. of LB Amount of Ag Sample ID used (mg) Broth(ml) in sample (mg) μg of Ag/mL SSD 20.00 5.00 6.05 1210.00 AgNO₃ 12.805.00 8.13 1626.00 AgNO₃ 25.00 5.00 15.90 3176.00 106/Tecophilic 11.305.00 0.73 146.00 (25:75) 106/Tecophilic 11.40 5.00 2.21 441.00 (75:25)SSD: silver sulfadiazine 1% cream

Thus, the antimicrobial activity of complex 106 was enhanced for alonger period, at a very low concentration of Ag particles byencapsulation in a suitable polymeric fiber. The bactericidal activityof the fiber mat 75% (complex 106/tecophilic) with 424 μg/mL of silveris 8 fold lower in the concentration of Ag than AgNO₃ (3176 μg/mL) andshowed not only a kill rate as fast as silver nitrate, but also retainedthe original color of the LB broth, a clear yellow solution unlikesilver nitrate which stains and changed the LB broth color to darkbrown. The silver-sulfadiazine cream did not readily dissolve in theaqueous LB broth, thus affecting the rate of its bactericidal activity.

The antimicrobial activity of the fiber mat encapsulating complex 106can be considered to be a combination of active silver species, whichmay include AgCl₂ ⁻ ions, clusters of Ag⁺ ions, AgCl and free Ag⁺ ions.Theoretically, the slow release of the active silver particles in thesolution leads to the quick formation of silver chloride. The presenceof more chloride anion as the major counter ion will further result inthe formation of negatively charged [Ag_(y)Cl_(x)]^(n−) ion species(where y=1, 2, 3 . . . etc; x=2, 3 . . . (y+1); n=x−1). The anionicsilver complexes of the type [AgI₃]²⁻, [Ag₂I₄]²⁻, [Ag₄I₈]⁴⁻ and[Ag₄I₆]²⁻ have been formed. The formation of anionic silver chloridespecies may not be limited to the leached aggregates of silver particlesin the solution, but may also be found on the surface of the fiber matsas shown in the SEM images of FIG. 30. Anionic silver dichloride isknown to be soluble in an aqueous media and thus will be bio-available.It has been reported that anionic silver halides are toxic to bothsensitive and resistance strain bacteria. The adsorbed active silverspecies on the network of fibers in the mat is an advantage the fibermat has to increase the surface area of the active silver species overthe conventional use of aqueous silver ions. This mechanism might haveaccounted for the effective bactericidal activity of the fiber mat in anaqueous media, even at such a low concentration of silver compared tothe un-encapsulated form of complex 106. Although complex 106 issparingly soluble in water, its quick decomposition has been observed tooccur in aqueous media. Thus, the bactericidal activity of complex 106is reduced due to poor availability of active silver species in the LBbroth media, which might be due to the formation of secondary silvercompound especially AgCl.

Acute Toxicity Assessment:

The LD 50 assessment was done by intravenous administration of 108,dissolved in a buffered saline solution, via the tail of rats. Adultrats were used with an average weight of 500 grams. Progressiveadministration of 0.3 mL of the dose (5 mg, 50 mg) was done weekly. Therats were carefully examined for the dose-response effect. Deathoccurred 10 minutes after administrating 50 mg of 108, when 50% of therats showed powerful convulsion before death. Autopsy report showedpulmonary hemorrhage and hemorrhage in the brain of the dead rats, adiagnosis of stroke. The surviving rats were observed to lose weight,with a drastic loss in appetite, and low urine out put. The LD 50assessment was found to be 100 mg/Kg of rat.

The synthesis of 108 with functionalized groups aids in tailoring theencapsulation of the silver(I) imidazole cyclophane gem diol into ananofiber. The fiber mat has been shown to have improved theantimicrobial activity of the silver(I)-n-heterocyclic carbene complexeson the inoculum, with a faster kill rate than silvadene in an LB brothmedium at a concentration 8 fold lower than silvadene. The encapsulationof the silver N-heterocyclic carbene complexes increases thebio-availability of active silver species and also reduces the amount ofsilver used. Encapsulated silver(I) carbene complexes in nano-fibers hasbeen demonstrated to be a promising material for sustained and effectivedelivery of silver ions over a longer period of time with maximumbactericidal activity than supplying silver in an aqueous form. Theamount of silver required for antimicrobial activity is reduced withthis technique of encapsulation compared to the un-encapsulated form,which often is related to the amount of silver in 0.5% silver nitrate.Furthermore, the ability of the fiber mat to retain the original colorof the LB broth is a major cosmetic plus. The assessment of the acutetoxicity of the ligand on rats showed an LD50 of 100 mg/Kg of rat, avalue considered to be moderately toxic.

In addition to useful antimicrobial, or antibacterial, properties, it isbelieved that the present invention can inhibit fungal growth, and alsoviral growth. The compositions of matter and methods of the presentinvention also contemplate delivery of silver to locations via any knownvehicle, including, but not limited to, inhalation through the lungs,direct application of a liquid to an eye, and direct application to aurinary bladder infection, or any other type of topical application.

General Experimental:

Silver (I) oxide, silver sulfadiazine and 1,3-dichloroacetone wherepurchased from Aldrich. Acetone, acetonitrile, methanol, ethanol,ammonium hexafluorophosphate, and organisms; S. cerevisiae (ATCC 2601),C. albicans (ATCC 10231), A. niger(ATCC 16404), E. coli (ATCC 8739), P.aeruginosa (ATCC 9027), S. aureus (ATCC 6538) were purchased fromFisher. All reagents were used without further purification. Infraredspectra were recorded on Nicolet Nexus 870 FT-IR spectrometer. The ¹Hand ¹³C NMR data was recorded on a Varian Gemini 300 MHz instrument, andthe spectra obtained were referenced to the deuterated solvents. Massspectroscopy data were recorded on an ESI-QIT Esquire-LC with a positiveion polarity. The TEM images were recorded on FEI TE CNAI-12transmission electron microscope (TEM) at 120 KV.

Synthesis of the Imidazolium Cyclophane Gem-Diol Dichloride:

A solution containing 0.24 grams (1.0 mmol) of2,6-bis(imidazolemethyl)pyridine and 0.254 grams (2.0 mmol)1,3-dichloroacetone in 60 mL of acetonitrile was stirred at 75° C. for 8hours to obtain 108 as a brown solid on filtration. The yield is 0.9mmol at a rate of 89.6%. Colorless crystals of the PF₆ salt of 108 wereobtained by slow evaporation from acetonitrile/water. The melting pointis 175 to 178° C. ¹H NMR (300 MHz, DMSO-d₆): δ 4.68 (s, 4H, CH ₂C(OH)₂CH₂), 5.67 (s, 4H, CH₂), 7.40, (s, 2H, NC(H)CH), 7.47 (d, 2H, J=7.8 Hz,m-pyr), 7.65 (s, 2H, C(OH), 7.89 (s, 2H, NCHC(H)), 7.94 (t, 1H, J=7.8Hz, p-pyr), 9.34 (s, 2H, NC(H)N). ¹³C NMR (75 MHz, DMSO-d₆): δ 51.8,55.2, 91.1, 120.5, 122.0, 123.9, 138.0, 138.8, 152.6. ESI-MS m/z: 384[M²⁺2Cl⁻], 348 [M²⁺Cl⁻]. FTIR (Nujol, cm⁻¹): 3387, 3105, 1597, 1564,1439, 1346, 1171, 1085, 996, 755. Anal. Calcd: C, 48.54; H, 4.41; N,16.94; Cl, 17.13. Found: C, 48.33; H, 4.32; N, 16.71; Cl, 16.76.

Synthesis of the Dinuclear Silver Carbene Cyclophane Gem-Diol Hydroxide:

The combination of 0.232 grams (1.0 mmol) silver (I) oxide and 0.366grams (0.9 mmol) of 108 in 70 mL methanol was stirred at roomtemperature for 50 minutes. The filtrate was concentrated to obtaincomplex 106 as a yellow solid. Single crystals of complex 106 wereobtained from ethanol, containing a spike of carbonate, by slowdiffusion.

Yield: 0.618 grams, 0.738 mmol, 82%. The melting point is 202 to 204° C.ESI-MS m/z: 400[0.5M²⁺], 801[2M⁺], 837[2M⁺2OH⁻]. FTIR (Nujol, cm⁻¹):3415, 3105, 1596, 1564, 1439, 1344, 1169, 1084, 1028, 996, 758. ¹³C NMR(75 MHz, DMSO-d₆): δ 48.6, 51.1, 53.8, 92.1, 119.9 (J=1.4 Hz), 121.6,128.6, 137.8 (J=2.4 Hz), 154.2, 184.9 (Jcarbene-Ag=211 Hz). Anal. Calcd:Ag, 24.54; C, 43.79; H, 4.20; N, 15.24. Found: C, 43.15; H, 4.22; N,14.89.

Electrospun Fiber:

Tecophilic® was dissolved in a mixture of ethanol and tetrahydrofuran ata ratio of 9 to 1. A solution of complex 106 in ethanol was mixed with apre-made solution of Tecophilic®. Solutions with different weight ratiosbetween complex 106 and Tecophilic® were prepared. The ratios were0/100, 25/75 and 75/25. The solutions of complex 106 and Tecophilic®were held in a pipette. An electrical potential difference of 15 KV wasapplied between the surfaces of the solution drop to the groundedcollector, a distance of about 20 cm. Transmission electron microscopy(TEM) and scanning electron microscopy (SEM) were used to characterizethe as-spun fibers and fibers exposed to water.

Antimicrobial Test:

Sterilized LB Broth was measured (5 mL) into a sterile tube. A loopfulof stationary phase cultured microorganism (E. coli, P. aeruginosa, S.aureus) was introduced into the tube containing the LB Broth solution.The mixture was cultured overnight, at 35° C. in a shaking incubator.The same procedure was done with stationary phased cultured fungi (C.albican, S. cerevisae, A. niger) and incubated without shaking at roomtemperature for 72 hours.

Fiber Mat Testing:

A constant volume (25 μL) of the freshly grown organism was placed on anLB agar plate and grown to obtain a lawn of the organism. A fiber mat(2.0 cm×2.0 cm) of complex 106 and pure Tecophilic was placed on a lawnof bacteria (E. coli, P. aeruginosa, S. aureus) of an LB agar plate andincubated overnight at 35° C. The bactericidal activity was observed byvisual inspection of growth and no growth in and around the area of thefiber mat. About the same dimension of the fiber mat was placed on alawn of fungi (C. albicans, S. cerevisiae, A. niger) and incubated atroom temperature for 48 hours. The diameter of the clear zone wasmeasured.

Minimum Inhibitory Concentration (MIC) Test:

Serial dilutions were made to obtain a range of concentrations bytransferring 1 mL of freshly prepared stock solution of the silvercompounds (with the same amount of silver particles) into a sterileculture tube containing 2 mL of LB broth, marked A. 1 mL of well mixedsolution of A was transferred to culture tube B containing LB broth. Thesame procedure was repeated to obtain the dilute solution for tube C, Dand E. The MIC was determined by visual inspection of growth/no-growthof the above concentrations of the silver compounds marked A-Einoculated with 25 μL of the organisms. After incubation at 35° C.overnight with no growth of organism, an additional 80 μL of freshlygrown organisms was added to each of the culture on the second day andincubated at the same temperature.

Kinetic Test of Bactericidal Activity:

Equal volume (5 mL) of LB broth were measured into sterile culture tubesand inoculated with 100 μL of S. aureus to each tube containing silvernitrate (12.8 mg, 25 mg), silver sulfadiazine (20 mg), 11.3 mg complex106/Tecophilic (25:75) and 11.4 mg complex 106/Tecophilic (75:25) fibermats. The mixtures were incubated at 35° C. and the bactericidalactivity was checked over a range of time by streaking one loopful ofeach mixture on an agar plate. The agar plate was then incubated at 37°C. overnight and the numbers of colonies of organism formed counted. Thesame procedure was repeated using 100 μL P. aeruginosa.

Animal Studies:

Male Sprague Dawley available from Harlan Sprague Dawley (Indianapolis,Ind.) adult rats (400 to 500 grams body weight) were housed in theuniversity of Akron animal facility. Temperature and humidity were heldconstant, and the light/dark cycle was 6.00 am-6.00 pm: light, 6.00pm-6.00 am: dark. Food available from Lab diet 5P00, Prolab, PMInutrition, Intl. (Bretwood, Mo.) and water were provided ad libitum.Animals were anesthetized with ether in order to inject the compoundinto the tail vein, using a 27 gauge syringe needle in a volume of 0.3mL sterile saline. The dosages for the ligand were 5 mg and 50 mg. Atthe end dosages of the experiment, animals were terminated and theliver, lung, kidney and heart tissues were removed and frozen at −70° C.Urine samples were collected daily for later examination of the compounddistribution. These studies were approved by the University of AkronInstitutional Animal Care and Use Committee (IACUC).

X-ray Crystallographic Structure Determination:

Crystal data and structure refinement parameters contained in thesupporting information. Crystals of 108 and complex 106 were each coatedin paraffin oil, mounted on kyro loop, and placed on a goniometer undera stream of nitrogen. X-ray data were collected at a temperature of 100K on a Brucker Apex CCD diffractometer using Mo Kα radiation (λ=0.71073Angstroms). Intensity data were integrated using SAINT software, and anempirical absorption correction was applied using SADABS. Structures 108and complex 106 were solved by direct methods and refined usingfull-matrix least square procedures. All non-hydrogen atoms were refinedwith anisotropic displacement.

Additional Embodiments:

In another embodiment, the present invention relates to metal complexesof N-heterocyclic carbenes that contain an anti-fungal and/oranti-microbial moiety and/or group in combination with one or moreadditional active moieties and/or groups selected from fluoroquinolonecompounds or derivatives thereof; steroids or derivatives thereof;anti-inflammatory compounds or derivatives thereof; anti-fungalcompounds or derivatives thereof; anti-bacterial compounds orderivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N. In still another embodiment, the present inventionrelates to metal complexes of N-heterocyclic carbenes that contain ananti-fungal and/or anti-microbial moiety and/or group in combinationwith two or more additional active moieties and/or groups selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N.

Such double, triple or higher action compounds can be represented by thecompounds represented by Formulas 301 to 305 shown below:

where R¹, R², R³, R⁴, R⁶ and R⁷, if present, are each independentlyselected from hydrogen; hydroxy; C₁ to C₁₂ alkyl; C₁ to C₁₂ substitutedalkyl; C₃ to C₁₂ cycloalkyl; C₃ to C₁₂ substituted cycloalkyl; C₂ to C₁₂alkenyl; C₃ to C₁₂ cycloalkenyl; C₃ to C₁₂ substituted cycloalkenyl; C₂to C₁₂ alkynyl; C₆ to C₁₂ aryl; C₅ to C₁₂ substituted aryl; C₆ to C₁₂arylalkyl; C₆ to C₁₂ alkylaryl; C₃ to C₁₂ heterocyclic; C₃ to C₁₂substituted heterocyclic; C₁ to C₁₂ alkoxy; C₁ to C₁₂ alcohols; C₁ toC₁₂ carboxy; biphenyl; C₁ to C₆ alkyl biphenyl; C₂ to C₆ alkenylbiphenyl; or C₂ to C₆ alkynyl biphenyl, and where R⁵ is selected fromfluoroquinolone compounds or derivatives thereof; steroids orderivatives thereof; anti-inflammatory compounds or derivatives thereof;anti-fungal compounds or derivatives thereof; anti-bacterial compoundsor derivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N.

In another embodiment, the compounds of the present invention are“triple action” compounds due to the inclusion of two active substituentgroups selected from fluoroquinolone compounds or derivatives thereof;steroids or derivatives thereof; anti-inflammatory compounds orderivatives thereof; anti-fungal compounds or derivatives thereof;anti-bacterial compounds or derivatives thereof; antagonist, compoundsor derivatives thereof; H₂ receptor compounds or derivatives thereof;chemotherapy compounds or derivatives thereof; tumor suppressorcompounds or derivatives thereof; or C₁ to C₁₆ alkyl heteroatom groupswhere the heterotatom is selected from S, O, or N. In this embodimentone of R¹, R², R³, R⁴, R⁶ or R⁷ is selected from fluoroquinolonecompounds or derivatives thereof; steroids or derivatives thereof;anti-inflammatory compounds or derivatives thereof; anti-fungalcompounds or derivatives thereof; anti-bacterial compounds orderivatives thereof; antagonist compounds or derivatives thereof; H₂receptor compounds or derivatives thereof; chemotherapy compounds orderivatives thereof; tumor suppressor compounds or derivatives thereof;or C₁ to C₁₆ alkyl heteroatom groups where the heterotatom is selectedfrom S, O, or N, where the remaining R¹, R², R³, R⁴, R⁶ and R⁷ groupsare each independently selected from hydrogen; hydroxy; C₁ to C₁₂ alkyl;C₁ to C₁₂ substituted alkyl; C₃ to C₁₂ cycloalkyl; C₃ to C₁₂ substitutedcycloalkyl; C₂ to C₁₂ alkenyl; C₃ to C₁₂ cycloalkenyl; C₃ to C₁₂substituted cycloalkenyl; C₂ to C₁₂ alkynyl; C₆ to C₁₂ aryl; C₅ to C₁₂substituted aryl; C₆ to C₁₂ arylalkyl; C₆ to C₁₂ alkylaryl; C₃ to C₁₂heterocyclic; C₃ to C₁₂ substituted heterocyclic; C₁ to C₁₂ alkoxy; C₁to C₁₂ alcohols; C₁ to C₁₂ carboxy; biphenyl; C₁ to C₆ alkyl biphenyl;C₂ to C₆ alkenyl biphenyl; or C₂ to C₆ alkynyl biphenyl, and where R⁵ isselected from fluoroquinolone compounds or derivatives thereof; steroidsor derivatives thereof; anti-inflammatory compounds or derivativesthereof; anti-fungal compounds or derivatives thereof; anti-bacterialcompounds or derivatives thereof; antagonist compounds or derivativesthereof; H₂ receptor compounds or derivatives thereof; chemotherapycompounds or derivatives thereof; tumor suppressor compounds orderivatives thereof; or C₁ to C₁₆ alkyl heteroatom groups where theheterotatom is selected from S, O, or N. In this embodiment, the twoactive substituent groups attached to Compounds 301 through 305 shouldnot be the same.

In one embodiment, the one or more double, triple or high actioncompounds of the present invention can contain one or more additionalactive groups, or moieties, as defined above that are bound directly toone or more points of the metal complexes of N-heterocyclic carbenesdisclosed herein. In this embodiment, no intervening linking group, orgroups, are needed. In another embodiment, suitable linking groups canbe utilized to bind the one or more additional active groups, ormoieties, to one or more points of the metal complexes of N-heterocycliccarbenes disclosed herein. Suitable linking groups are known in the artand as such a discussion thereof is omitted for the sake of brevity.

One example of one of the above compounds is shown in FIG. 31 amultifunctional SCC of Formula 301 where R¹ through R⁴ are comprised ofmethyl groups and the R⁵ carboxylate is ibuprofen. The synthesis of SCCIIBU is achieved by using one equivalent of 1,3,7,9-tetramethylxanthiniumiodide and two equivalents of silver (I) ibuprofen salt were stirred inmethanol. After 1.5 hours a yellow precipitate was filtered from thereaction mixture through Celite®. The filtrate was collected andvolatiles removed via rotary evaporation. The crude solid was stirred indiethyl ether to afford SCCI IBU as a white solid.

TABLE 7 Efficacy Data Formula 301 Formula 301 R⁵ = CH₃ R⁵ = ibuprofenTobramycin Bacterial species SCC1 (μg/ml) SCC1-IBU (μg/ml) (μg/ml)Isolate MIC MBC MIC MBC MIC MBC Pseudomonas aeruginosa PA 01-V 1 2 2 615 >20 PA M57-15 2 4 1 2 0.5 1 PA RR05 0.5 1 0.25 2 0.5 1 PA HP3 4 80.25 2 4 8 PA LF05 1 1 1 1 0.5 1 Alcaligenes xylosoxidans AX 22 0.5 20.5 2 20 >20 AX RE05 4 8 1 2 >20 >20 Stenotrophomonas maltophilia SMAH08 4 8 1 2 8 20 Methicillin resistant Staphylococcus aureus (MRSA) SALL06 8 10 2 6 >20 >20 SA EH06 6 20 2 4 >20 >20 Yersinia pestisYP1-1CO92- 0.5 1 0.5 2 0.25 0.25 LCR- Escherichia coli J53 1 1 0.5 0.5 11 J53 + pMG101 >20 >20 >20 >20 1 1

It should be evident that the present invention is highly effective inproviding a method of inhibiting microbial growth by administration of aN-functionalized silver carbene complex. It is, therefore, to beunderstood that any variations evident fall within the scope of theclaimed invention and thus, the selection of specific component elementscan be determined without departing from the spirit of the inventionherein disclosed and described.

What is claimed is:
 1. A method for treating a bacterial infectioncomprising the step of administering to a patient in need thereof atherapeutically effective amount of a compound selected from the groupconsisting of